Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET

The similar to 15 kDa variable domains of camelid heavy-chain-only antibodies (called NanobodiesA (R)) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with Ga-68 via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for Zr-89 immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified Ga-68 was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml(-1) gentisic acid, pH 5.5) at 4A degrees C or in human serum at 37A degrees C, a mixture of Ga-67 and Ga-68 was used. Biodistribution and immuno-PET studies of Ga-68-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using Zr-89-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall Ga-68 radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was > 96%, while the integrity and immunoreactivity were preserved. Ga-68/67-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (< 2% radioactivity loss). In biodistribution studies the Ga-68-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 +/- 1.3 to 7.2 +/- 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate Zr-89-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. Via a rapid procedure under mild conditions a Ga-68-Nanobody was obtained that exhibited high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts. Fast kinetic Ga-68-Nanobody conjugates can be promising tools for tumour detection and imaging of target expression.